Compounds and compositions as itpkb inhibitors

ABSTRACT

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with abnormal or dysregulated B cell activities, particularly diseases or disorders that involve aberrant activation of inositol 1,4,5-trisphosphate 3-kinase B (ITPKb).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/944,345, filed Jun. 15, 2007, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention provides a novel class of compounds, pharmaceuticalcompositions comprising such compounds and methods of using suchcompounds to treat or prevent diseases or disorders associated withabnormal or dysregulated B cell activities, particularly diseases ordisorders that involve aberrant activation of inositol1,4,5-trisphosphate 3-kinase B (ITPKb).

BACKGROUND

The protein kinases represent a large family of proteins, which play acentral role in the regulation of a wide variety of cellular processesand maintaining control over cellular function. A partial, non-limiting,list of these kinases include: non-protein substrate kinases such asIPTKb; receptor tyrosine kinases such as platelet-derived growth factorreceptor kinase (PDGF-R), the nerve growth factor receptor, trkB, Met,and the fibroblast growth factor receptor, FGFR3; non-receptor tyrosinekinases such Abl and the fusion kinase BCR-Abl, Lck, Csk, Fes, Bmx andc-src; and serine/threonine kinases such as b-RAF, c-RAF, sgk, MAPkinases (e.g., MKK4, MKK6, etc.) and SAPK2a, SAPK2β and SAPK3. Aberrantkinase activity has been observed in many disease states includingbenign and malignant proliferative disorders as well as diseasesresulting from inappropriate activation of the immune and nervoussystems.

The novel compounds of this invention inhibit the activity of ITPKb andare, therefore, expected to be useful in the treatment ofITPKb-associated diseases.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I:

in which:

-   -   n is selected from 0, 1, 2 and 3;    -   m is selected from 0, 1, 2 and 3;    -   A can have up to 3 groups selected from —CR₁═, —CR₂═ and —CR₅═        replaced with —N═;    -   R₁, R₂ and R₅ are independently selected from hydrogen, hydroxy,        halo, cyano, C₁₋₆alkyl, halo-substituted-C₁₋₆alkyl,        hydroxy-substituted-C₁₋₆alkyl and cyano-substituted-C₁₋₆alkyl;    -   R₃ and R₄, together with the carbon atoms to which R₃ and R₄ are        attached, form a 5 to 6 member heterocycle fused to ring A        containing up to 4 radicals selected from O, C(O), S(O)₂,        CR₁₁R₁₂ and NH; wherein each R₁₁ and R₁₂ are independently        selected from hydrogen, C₁₋₃alkyl, and        halo-substituted-C₁₋₃alkyl; or R₁₁ and R₁₂, together with the        carbon to which they are both attached, forms C₃₋₇cycloalkyl;    -   R₆ and R₇ are independently selected from hydrogen, C₁₋₃alkyl        and halo-substituted-C₁₋₃alkyl; or R₆ and R₇, together with the        carbon to which they are both attached, forms C₃₋₇cycloalkyl;    -   R₈ is selected from C₁₋₆alkyl, C₂₋₆alkenyl,        halo-substituted-C₁₋₆alkyl and hydroxy-substituted-C₁₋₆alkyl; or        two R₈ groups can combine to form an alkyl bridge; or when two        R₈ groups are attached to the same carbon atom, they, together        with the carbon to which they are both attached, form        C₃₋₇cycloalkyl;    -   R₉ is selected from L₁-C₆₋₁₀aryl, L₁-C₁₋₁₀heteroaryl, C₁₋₆alkyl,        L₁-C₃₋₁₂cycloalkyl and L₁-C₃₋₈heterocycloalkyl; wherein said        aryl, heteroaryl, cycloalkyl and heterocycloalkyl of R₉ can be        optionally substituted with 1 to 3 radicals independently        selected from halo, cyano, hydroxy, C₁₋₃alkyl,        halo-substituted-C₁₋₃alkyl, cyano-substituted-C₁₋₃alkyl,        hydroxy-substituted-C₁₋₃alkyl, —C(O)R₁₃, —C(O)NR₁₃R₁₄; wherein        each R₁₃ and R₁₄ are independently selected from hydrogen and        C₁₋₆alkyl;    -   L₁ is a bond, C₁₋₃alkyl or halo-substituted-C₁₋₃alkyl;    -   Y is N or CR₁₀;    -   R₁₀ is selected from hydrogen, C₁₋₆alkyl, —NR₁₅R₁₆, —NR₁₅C(O)R₁₆        and —C(O)NR₁₅R₁₆; wherein each R₁₅ and R₁₆ are independently        selected from hydrogen, C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₁₀heteroaryl,        C₃₋₁₂cycloalkyl and C₃₋₈heterocycloalkyl; wherein said aryl,        heteroaryl, cycloalkyl and heterocycloalkyl can be optionally        substituted with 1 to 3 radicals independently selected from        halo, hydroxy, cyano, C₁₋₆alkyl, halo-substituted-C₁₋₆alkyl,        C₁₋₆alkoxy and halo-substituted-C₁₋₆alkoxy; and the N-oxide        derivatives, prodrug derivatives, protected derivatives,        individual isomers and mixture of isomers thereof;    -   and the pharmaceutically acceptable salts and solvates (e.g.        hydrates) of such compounds.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, individual isomers and mixture of isomers thereof; or apharmaceutically acceptable salt thereof, in admixture with one or moresuitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which inhibition of kinase activity,particularly ITPKb activity, can prevent, inhibit or ameliorate thepathology and/or symptomology of the diseases, which method comprisesadministering to the animal a therapeutically effective amount of acompound of Formula I or a N-oxide derivative, individual isomers andmixture of isomers thereof, or a pharmaceutically acceptable saltthereof.

In a fourth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which kinase activity, particularly ITPKb activity,contributes to the pathology and/or symptomology of the disease.

In a fifth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Alkyl” as a group and as a structural element of other groups, forexample halo-substituted-alkyl and alkoxy, can be eitherstraight-chained or branched. C₁₋₄-alkoxy includes, methoxy, ethoxy, andthe like. Halo-substituted alkyl includes trifluoromethyl,pentafluoroethyl, and the like. Hydroxy-substituted alkyl includeshydroxy-methyl, hydroxy-ethyl and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, aryl may be phenylor naphthyl, preferably phenyl. “Arylene” means a divalent radicalderived from an aryl group.

“Heteroaryl” is as defined for aryl above where one or more of the ringmembers can be a heteroatom selected from —O—, —N═, —NR—, —C(O)—, —S—,—S(O)— or —S(O)₂—, wherein R is hydrogen, C₁₋₄alkyl or a nitrogenprotecting group. For example C₁₋₁₀heteroaryl includes pyridyl, indolyl,indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,benzothiopyranyl, benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl,pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl,pyrazolyl, thienyl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁₋₄alkyl or a nitrogen protecting group. Forexample, C₃₋₈heterocycloalkyl as used in this application to describecompounds of the invention includes morpholino, pyrrolidinyl,pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

“Treat”, “treating” and “treatment” refer to a method of alleviating orabating a disease and/or its attendant symptoms.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compounds, compositions and methods forthe treatment of kinase related disease, particularly IPTKb relateddiseases. For example, autoimmune diseases, particularly B cellassociated diseases, are related to IPTKb. For example, rheumatoidarthritis, systemic lupus erythematosus (SLE), immune thrombocytopenicpurpura (ITP) and hemolytic anemia.

In one embodiment, with reference to compounds of Formula I, n isselected from 1 and 2; m is selected from 0, 1 and 2; A can have up to 3groups selected from —CR₁═, —CR₂═ and —CR₅═ replaced with —N═; R₁, R₂and R₅ are hydrogen; R₆ and R₇ are hydrogen; R₈ is selected fromC₁₋₆alkyl, halo-substituted-C₁₋₆alkyl and hydroxy-substituted-C₁₋₆alkyl;or two R₈ groups can combine to form an alkyl bridge; or when two R₈groups are attached to the same carbon, they, together with the carbonto which they are both attached, form C₃₋₇cycloalkyl; R₉ is selectedfrom L₁-C₆₋₁₀aryl, L₁-C₁₋₁₀heteroaryl, C₁₋₆alkyl, L₁-C₃₋₁₂cycloalkyl andL₁-C₃₋₈heterocycloalkyl; wherein said aryl, heteroaryl, cycloalkyl andheterocycloalkyl of R₉ can be optionally substituted with 1 to 3radicals independently selected from halo, cyano, hydroxy, C₁₋₃alkyl,halo-substituted-C₁₋₃alkyl, cyano-substituted-C₁₋₃alkyl,hydroxy-substituted-C₁₋₃alkyl, —C(O)R₁₃, —C(O)NR₁₃R₁₄; wherein each R₁₃and R₁₄ are independently selected from hydrogen and C₁₋₆alkyl; L₁ is abond or C₁₋₃alkyl; Y is CR₁₀, and R₁₀ is hydrogen.

In another embodiment, the 5 to 6 member heterocycle fused to ring Aformed from R₃ and R₄, together with the carbon atoms to which R₃ and R₄are attached, is selected from:

In another embodiment, R₈ is selected from methyl, ethyl,trifluoromethyl, difluoromethyl, fluoromethyl and hydroxy-methyl; or twoR₈ groups can combine to form an alkyl bridge selected from methyl,ethyl and propyl; or two R₈ groups are attached to the same carbon,they, together with the carbon to which they are both attached, formcyclopropyl.

In another embodiment, R₉ is selected from C₃₋₇cycloalkyl,C₄₋₇heterocycloalkyl, phenyl, pyridinyl, pyrazinyl, pyrimidinyl andfuro[3,2-c]pyridin-4-yl; wherein said phenyl, pyridinyl, pyrazinyl,pyrimidinyl or furo[3,2-c]pyridin-4-ylis optionally substituted with 1to 3 radicals independently selected from trifluoromethyl, cyano, bromo,chloro, hydroxy-methyl, methyl-carbonyl, methyl, amino-carbonyl, nitro,iodo, fluoro, methoxy-carbonyl, hydroxy, amino, carboxy and methoxy.

In another embodiment, are compounds selected from:6-{4-[4-(5-Trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyrazol-3-yl}-benzo[e][1,3]oxazine-2,4-dione;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((S)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;7-(4-4(R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)isoquinoline-1,3(2H,4H)-dione;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;7-(4-((4(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;and6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-5-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;6-(4-((4(3-(5-(trifluoromethyl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((2-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4(3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;(S)-6-(4-((3-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[1)][1,4]oxazin-3(4H)-one;6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;(S)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-2-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)isonicotinonitrile;(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(5-methylpyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(54(3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-4,4-dimethyl-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-44-(5-(trifluoromethyl)pyridin-2-yl)-4,7-diazaspiro[2.5]octan-7-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(2-methyl-4-43-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)nicotinonitrile;(R)-6-(4-44-(5-chloropyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-1-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-2-carboxylicacid;(S)-6-(4-((2-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((2-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-44-(2-fluorobenzyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((4-(4-chlorophenyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one,(R)-6-(4-((3-methyl-4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one,and(S)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one.

Further compounds of the invention are detailed in the Examples andTable I, infra.

Pharmacology and Utility

Compounds of the invention modulate the activity of IPTKb and, as such,are useful for treating diseases or disorders in which aberrant activityof IPTKb, contributes to the pathology and/or symptomology of diseases.

By inhibiting B cell activation and development, the ITPKb inhibitors ofthe present invention are useful in various therapeutic applications.Pharmacological inhibition of ITPKb provides a means to inhibit B cellmalfunction in pathological settings. For example, B cells play apathological role in chronic transplant rejection, and the developmentof autoimmune diseases (e.g. Rheumatoid Arthritis, SLE, lupus, and thelike), Psoriasis, Allergy (Asthma, Rhinitis, COPD, Dermatitis) andothers, including anaphylaxis and many complement mediated diseases. TheITPKb-inhibiting compounds of the invention can be effective agents totreat these diseases where ITPKb acts to promote pathogenesis.

Other diseases and conditions that are amenable to treatment includediseases associated with or mediated by abnormal B cell proliferation,e.g., B cell lymphoma. They also encompass other antibody-mediateddisorders, e.g., allergies, psoriasis, systematic lupus erythematosus(SLE), primary binary cirrhosis (PBC), and idiopathic thrombocytopenicpurpura (ITP). In addition to treating these diseases or conditions,ITPKb inhibitors of the present invention are also useful for preventingor modulating the development of such diseases or disorders in a subject(including human and animals such as other mammals) suspected of being,or known to be, prone to such diseases or disorders. The B-cellmodulators that can be employed in the therapeutic applications of theinvention include the specific ITPKb-inhibitors described in theExamples and tables, infra.

The invention thus provides a method for modulating B lymphocytedevelopment and function in a subject (human or other mammal) for thetreatment of autoimmune diseases, the method comprising administering tothe subject a compound of formula I or a pharmaceutical compositionthereof in an effective amount to modulate the kinase activity orcellular level of ITPKb (such as demonstrated by the in vitro assaysdescribed, infra); thereby modulating B lymphocyte differentiation andfunction in a subject. The compound can down-regulate the cellular levelof the ITPKb molecule by inhibiting the kinase activity of ITPKb.

In accordance with the foregoing, the present invention further providesa method for preventing, treating and/or ameliorating the condition ofany of the diseases or disorders described above in a subject in need ofsuch treatment, which method comprises administering to said subject atherapeutically effective amount (See, “Administration andPharmaceutical Compositions”, infra) of a compound of Formula I or apharmaceutically acceptable salt thereof. Compounds of Formula I candown-regulate the cellular level of the ITPKb molecule by inhibiting thekinase activity of ITPKb such as described by the in vitro assaysdescribed, infra. For any of the above uses, the required dosage willvary depending on the mode of administration, the particular conditionto be treated and the effect desired.

Administration and Pharmaceutical Compositions

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors. Ingeneral, satisfactory results are indicated to be obtained systemicallyat daily dosages of from about 0.03 to 2.5 mg/kg per body weight. Anindicated daily dosage in the larger mammal, e.g. humans, is in therange from about 0.5 mg to about 100 mg, conveniently administered, e.g.in divided doses up to four times a day or in retard form. Suitable unitdosage forms for oral administration comprise from ca. 1 to 50 mg activeingredient.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, e.g.,orally, e.g., in the form of tablets or capsules, or parenterally, e.g.,in the form of injectable solutions or suspensions, topically, e.g., inthe form of lotions, gels, ointments or creams, or in a nasal orsuppository form. Pharmaceutical compositions comprising a compound ofthe present invention in free form or in a pharmaceutically acceptablesalt form in association with at least one pharmaceutically acceptablecarrier or diluent can be manufactured in a conventional manner bymixing, granulating or coating methods. For example, oral compositionscan be tablets or gelatin capsules comprising the active ingredienttogether with a) diluents, e.g., lactose, dextrose, sucrose, mannitol,sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol;for tablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

Compounds of the invention can be administered in therapeuticallyeffective amounts in combination with one or more therapeutic agents(pharmaceutical combinations). For example, synergistic effects canoccur with other immunomodulatory or anti-inflammatory substances, forexample when used in combination with cyclosporin, rapamycin, orascomycin, or immunosuppressant analogues thereof, for examplecyclosporin A (CsA), cyclosporin G, FK-506, rapamycin, or comparablecompounds, corticosteroids, cyclophosphamide, azathioprine,methotrexate, brequinar, leflunomide, mizoribine, mycophenolic acid,mycophenolate mofetil, 15-deoxyspergualin, immunosuppressant antibodies,especially monoclonal antibodies for leukocyte receptors, for exampleMHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, orother immunomodulatory compounds, such as CTLA41 g. Where the compoundsof the invention are administered in conjunction with other therapies,dosages of the co-administered compounds will of course vary dependingon the type of co-drug employed, on the specific drug employed, on thecondition being treated and so forth.

The invention also provides for a pharmaceutical combinations, e.g. akit, comprising a) a first agent which is a compound of the invention asdisclosed herein, in free form or in pharmaceutically acceptable saltform, and b) at least one co-agent. The kit can comprise instructionsfor its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of Formula I and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T.W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I, wherein R₆ and R₇ are both hydrogen, can beprepared by proceeding as in the following Reaction Scheme I:

in which n, m, A, R₁, R₂, R₃, R₄, R₅, R₈, R₉ and R₁₀ are as defined inthe Summary of the Invention.

A compound of Formula I can be prepared by reacting of a compound offormula 3 with a compound of formula 4 in the presence of a suitablesolvent (e.g., DCM) using an appropriate reducing agents (e.g.,NaCNBH₃). A compound of formula 3 can be prepared by reacting of acompound formula 2 with the complex of POCl₃ and DMF followed by theaddition of a suitable base (e.g., NaOH).

Detailed examples of the synthesis of a compound of Formula I can befound in the Examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) that of reaction scheme I; and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

The present invention also includes all suitable isotopic variations ofthe compounds of the invention, or pharmaceutically acceptable saltsthereof. An isotopic variation of a compound of the invention or apharmaceutically acceptable salt thereof is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that may be incorporated into the compounds of theinvention and pharmaceutically acceptable salts thereof include but arenot limited to isotopes of hydrogen, carbon, nitrogen and oxygen such asas ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸O, ³⁵S, ¹⁸O, ³⁵S, ¹⁸F,³⁶Cl and ¹²³I. Certain isotopic variations of the compounds of theinvention and pharmaceutically acceptable salts thereof, for example,those in which a radioactive isotope such as ³H or ¹⁴C is incorporated,are useful in drug and/or substrate tissue distribution studies. Inparticular examples, ³H and ¹⁴C isotopes may be used for their ease ofpreparation and detectability. In other examples, substitution withisotopes such as may may afford certain therapeutic advantages resultingfrom greater metabolic stability, such as increased in vivo half-life orreduced dosage requirements. Isotopic variations of the compounds of theinvention or pharmaceutically acceptable salts thereof can generally beprepared by conventional procedures using appropriate isotopicvariations of suitable reagents. Isotopic variations of the compoundshave the potential to change a compound's metabolic fate and/or createsmall changes in physical properties such as hydrophobicity, and thelike. Isotopic variation have the potential to enhance efficacy andsafety, enhance bioavailability and half-life, alter protein binding,change biodistribution, increase the proportion of active metabolitesand/or decrease the formation of reactive or toxic metabolites.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The present invention is further exemplified, but not limited, by thefollowing examples that illustrate the preparation of compounds ofFormula I according to the invention.

Example 16-{4-[4-(5-Trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyrazol-3-yl}-benzo[e][1,3]oxazine-2,4-dione

A mixture of 2-chloro-5-trifluoromethylpyridine (362 mg, 1.99 mmol) and[1,4]-diazepane (1000 mg, 9.98 mmol) in DMF (6.0 mL) is stirred for 2hours at room temperature followed by evaporation of the solvent DMFunder vacuo. The residue is distributed between ethyl acetate (30 mL)and water (40 mL). The aqueous phase is extracted once with ethylacetate (20 m) and the combined organic phases are dried with Na₂SO₄.Evaporation gives an oily residue (crude1-(5-trifluoromethyl-pyridin-2-yl)[1,4]-diazepane) which is useddirectly for reductive amination in the last step.

To the suspension of 5-acetylsalicylamide (900 mg, 5.02 mmol) inpyridine (3.5 mL) at 0° C. under stirring is added ethylchloro-carbamate (600 mg, 5.53 mmol) dropwise and the reaction is heatedat 98° C. overnight. Pyridine is evaporated and the residue isdistributed between ethyl acetate (100 mL) and water (60 mL). Theorganic phase is washed with HCl (2N, 50 mL) and water (60 mL) and driedwith Na₂SO₄. As the organic phase is evaporated, the product6-acetyl-benzo[e][1,3]oxazine-2,4-dione starts to precipitate. Theprecipitate is collected by filtration and then further concentrateduntil the starting material (5-acetylsalicylamide) begins toco-crystallize.

To a suspension of 6-acetyl-benzo[e][1,3]oxazine-2,4-dione (626 mg, 3.05mmol) obtained above in ethanol (5.0 mL), is added a solution ofNaOAc.3H₂O (910 mg, 2.2 equiv.) and semicarbazide hydrochloride (408 mg,1.20 equiv.) in water (5.0 mL). The mixture is heated at 92° C. for 2hours. After cooling to room temperature, the product is collected byfiltration, washed with acetonitrile and dried under vacuum to give thecorresponding semicarbazone derivative.

To anhydrous DMF (2.50 mL) at 0° C. under argon is added POCl₃ (0.60 mL,1.01 g, 6.56 mmol) dropwise. After 10 minutes, the ice-bath is removedto allow the temperature to rise to room temperature. The semicarbazoneobtained above is added in portions and the temperature is raised to andkept at 68° C. for 1 hour. The reaction is quenched into ice/water.Na₂CO₃ is used to raise the pH to about 11˜12 followed by neutralizationwith NH₄Cl saturated aqueous solution. The precipitate is collected byfiltration and dried in vacuum to give3-(2,4-dioxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazole-4-carbaldehyde.

The mixture of3-(2,4-dioxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazole-4-carbaldehyde(51.4 mg, 0.2 mmol) and1-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepane (24.5 mg, 0.1 mmol) inmethylene chloride (1.0 mL), methanol (0.5 mL) and acetic acid (15 μL)is stirred for 30 minutes at room temperature. Then Na(AcO)₃BH (84 mg,4.0 equiv.) is added and the reaction is stirred overnight at roomtemperature. After evaporation, the residue is redissolved in DMF (1.0mL) which is subject to reverse-phase preparative LC-MS(acetonitrile/water/TFA gradient 10-90% CH₃CN in 7.5 min, Ultro 120 5 uMC18Q, 75×30 mmID). The collected water/MeCN solution of the TFA salt ofthe product is evaporated to remove the acetonitrile. A saturatedaqueous solution of NaHCO₃ is added to raise the pH to about 8-9. Thenethyl acetate is used to extract the product and the organic phase isdried with Na₂SO₄. Evaporation of the solvent yields the free-based6-{4-[4-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyrazol-3-yl}-benzo[e][1,3]oxazine-2,4-dione.

Example 26-(5-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl]methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one

Example 2-1 Preparation of 2-(aminomethyl)-4-bromophenol

5-bromo-2-hydroxybenzamide (2.16 g, 10 mol) and NaBH₄ (1.52 g, 40 mol)are dissolved in dry THF (150 ml) in a two-neck septum cappedround-bottom flask. Iodine (5.06 g, 20 mol)) in dry THF is added undernitrogen atmosphere at 0° C. over 2.5 hours. The reaction mixture isrefluxed for 3 hours and then cooled to 0° C. The excess hydride isdestroyed by careful addition of 1N HCl. After removing most organicsolvent by vacuum, the acidic aqueous solution is diluted in 1N HCl (150ml) and washed three times with ether (30 ml each time). The pH of theaqueous solution is adjusted to pH=6-7 by careful addition of sodiumbicarbonate solid. The product is collected by filtration and thenwashed with water. The product is further dried in vacuum oven overnightgiving 2-(aminomethyl)-4-bromophenol which is used directly in the nextstep without further purification.

Example 2-2 Preparation of 6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one

A solution of 2-(aminomethyl)-4-bromophenol (I) (1.16 g, 5.7 mmol),triethylamthylamine (III) and carbonyldiimidazole (II) (1.86 g, 11.5mmol) in anhydrous THF (100 ml) is refluxed for 4 hours. The solvent isevaporated and the residue dissolved in dichloromethane (DCM) (100 ml).The organic solution is washed with 1N HCl (20 ml×3) and then with brine(50 ml). The solvent is then evaporated and the resulting residue ispurified by chromatography to give6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one as a white powder.

Example 2-3 Preparation of3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-one

To a solution of 6-bromo-3,4-dihydrobenzo[e][1,3]oxazin-2-one (114 mg,0.5 mmol), PdCl₂(PPh₃)₂ (60 mg, 0.05 mmol), CuI (20 mg, 0.1 mmol),tetrabutylammonium iodide (74 mg, 0.2 mmol) in 1 mL of DMF are addedprop-2-yn-1-ol (150 mg, 2.5 mmol) and triethylamine (0.8 mL) via syringeunder nitrogen atmosphere. The resultant mixture is heated at 75° C.until the aryl bromide is consumed, as monitored by TLC. The cooledsolution is partitioned between ethyl acetate and water, and the organiclayer is then washed with brine, and dried over Na₂SO₄. Evaporation ofthe solution followed by column chromatography afforded the couplingproduct: 3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-oneas colorless oil. m/e: 204 (M+1)

Example 2-4 Preparation of5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-4-carbaldehyde

3,4-dihydro-6-(3-hydroxyprop-1-ynyl)benzo[e][1,3]oxazin-2-one (30 mg,0.15 mmol) is dissolved in dimethyl formamide (DMF) (2 ml) and istreated with Dess-Martin reagent (130 mg, 0.3 mmol) at room temperature.After stirring for 1 hour, the reaction is quenched with 1N HCl (10 ml)and then extracted with ethyl acetate (EtOAc) (10 ml x5). The combinedorganic phase is washed with water, then saturated aqueous ammoniachloride, and then brine. After drying over sodium sulfate, the solventis removed by vacuum to give crude aldehyde which is used directly inthe next step.

The crude aldehyde obtained above is dissolved in DMSO (2 ml) and thentreated with sodium azide (98 mg, 1.5 mmol) at room temperature. Afterstirring for 4 hours, the reaction is quenched with aqueous sodiumbicarbonate solution (10 ml) and then extracted with EtOAc (10 ml x5).The combined organic phase is washed with water, then saturated aqueoussodium bicarbonate, and then brine. After drying over sodium sulfate,the solvent is removed by vacuum to give the crude product which is thenpurified by flash chromatography yeilding5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-4-carbaldehydeas a colorless oil. m/e: 245 (M+1).

Example 2-5 Preparation of6-(54(R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one

5-(3,4-dihydro-2-oxo-2H-benzo[e][1,3]oxazin-6-yl)-2H-1,2,3-triazole-4-carbaldehyde(15 mg, 0.06 mmol) and(R)-1-(5-(trifluoromethyl)pyridin-2-yl)-2-methylpiperazine (20 mg, 0.08mmol) are mixed in DMF (1 ml) followed by addition of acetic acid (AcOH)(30u1). The mizture is stirred at room temperature for 30 minute andthen sodium triacetate boron hydride (42 mg, 0.2 mmol) is added. Theresulting suspension is then stirred at room temperature overnight. Thereaction is quenched using an aqueous saturated solution of sodiumbicarbonate and then extracted with ethyl acetate (10 ml×4). Thecombined organic phase is concentrated and the resulting crude productis purified by HPLC giving6-(5-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one as awhite powder. m/e:474 (M+1).

By repeating the procedures described in the above example, usingappropriate starting materials, the following compounds of Formula I, asidentified in Table 1, are obtained.

TABLE 1 Physical Data Compound MS (m/z): Number Structure (M + 1) 2

473.2 3

487.2 4

487.2 5

473.2 6

459.2 7

527.2 8

459.2 9

459.2 10

471.2 11

527.2 12

485.2 13

499.2 14

491.2 15

432.2 16

459.2 17

404.2 18

527.2 19

527.2 20

489.2 21

22

23

24

25

26

27

430.2 28

474.2 29

419.2 30

473.2 31

410.3 32

474.2 33

501.2 34

485.2 35

430.2 36

439.2 37

503.2 38

489.2 39

491.2 40

472.2 41

436.2 42

438.2 43

472.2 44

473.2 45

459.2

Assays

Compounds of the present invention are assayed to measure their capacityto inhibit ITPKb according to the following assays:

Purification of ITPKb: The DNA sequence encoding murine ITPKb residues640-942 is amplified from a full-length construct in mammalianexpression vector pKDNZ by PCR. The 3′-primer incorporates a stop codonand an overhanging Pad site. The product is digested with Pad beforebeing ligated into the MH4 plasmid which has been prepared by digestionwith PmlI and PacI. Cloning into the MH4 plasmid adds the sequenceMGSDKIHHHHHH to the N-terminus of the translated region. Mutant enzymesare made by site-directed mutagenesis using the Stratagene Quikchangekit.

ITPKb is expressed in the HK100 strain of Escherichia coli. Typically, a4 L batch of cells is grown in LB with 0.1 μg/mL ampicillin to 0.5A₆₀₀at 30 degrees C., before induction with 0.02% L-arabinose for 6 hours.Cells are harvested by centrifugation, and pellets are resuspended in 50mL of 50 mM Tris (pH 8), 100 mM NaCl, 1 mM TCEP, and 0.1 mg/mL lysozyme,with 1 Complete protease inhibitor tablet (Roche). Cells are disruptedby sonication, and debris is removed by centrifugation for 40 minutes at35000 g.

Initial purification is performed using three nickel-Sepharose Hi-TrapHP 1 mL columns (Amersham) connected in series. After application of thepellet supernatants, the bound material is washed with 20 mM Tris (pH8.0), 20 mM imidazole, 10% glycerol (v/v), and 1 mM TCEP before elutionwith an imidazole gradient up to 200 mM.

Fractions containing ITPKb are identified by SDS-PAGE, and the purefractions ae concentrated and buffer exchanged using centriprep 20 15kDa columns into 20 mM Tris (pH 8), 200 mM KCl, 5 mM MgCl₂, 0.5 mM DTT,10% glycerol, 1 μM IP₃, and 20 μM ATP to a final protein concentrationof 7 mg/mL.

Biochemical Measurement of ITPKb Activity: ITPKb activity is determinedusing the Kinase-Glo (Promega) ATP depletion assay. The assay reactionbuffer consists of 50 mM Tris (pH 8.0), 100 mM NaCl, 1 mM DTT, 10%glycerol, 5 mM MgCl₂, 1 μM ATP, and 10 μM IP₃ (Alexis Biochemicals). 50nl of inhibitor is then added to each 40 μL reaction followed by a 10 μLaddition of purified ITPKb (final concentration of 60 nM). The reactionmixture is incubated for 60 minutes at room temperature and stopped bythe addition of an equal volume of kinase-glo reagent (Promega).Luminescence is measured using a Molecular Devices Acquest instrument.

Compounds of Formula I preferably have an IC₅₀ of less than 500 nM,preferably less than 250 nM, more preferably less than 100 nM atinhibiting the phosphorylation of IP3.

Measuring Intracellular IP3, IP4, and IP5 levels by HPLC: Jurkat cellsare obtained from ATCC (clone E6-1) (www.ATCC.org Cat#TIB-152). 10⁷cells in 1 ml of inositol free RPMI-1640 w/o serum, are pulse labeled at37° C. for 6 hours with 15 uCi of 3H myo-inositol in inositol. Cells arethen diluted to 4 ml of RPMI-1640 with 10% FBS and incubated overnightat 37° C. Cells are then concentrated and resuspended in 1 ml ofRPMI-1640 w/10% FBS. 1 μl of inhibitor in DMSO is then added. 50 μg ofOKT3 and 10 μg of anti-human CD28 (BD Pharmingen clone CD28.2) is addedfollowed by a 5 minute incubation at 37° C. Cells are then concentratedand the reaction quenched with the resuspension of the cell pellet in100 μL of PBS w/350 mM HCl. Extracts are then spun to remove proteinsand cellular debris. Labeled inositol polyphosphates in the extracts arethen resolved by HPLC on a Partisphere SAX column (15 cm×4.6 mm).Samples are eluted as follows with gradients generated by mixing bufferA (10 mM (NH₄)H₂PO₄, pH 3.35, with H₃PO₄) with buffer B (1.7 M(NH₄)H₂PO₄, pH 3.35, with H₃PO₄). 0-12.5 minutes 0-100% Buffer B;12-5-25 minutes 100% Buffer B; 25-30 minutes 0-100% buffer A; 30-45minutes 100% buffer A. Radioactivity is detected with an online β-Ramdetector from IN/US systems.

Compounds of Formula I preferably have an IC₅₀ of less than 1 μM, morepreferably less than 500 nM in inhibiting the conversion of IP3 to IP4.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A compound of Formula I:

wherein: n is selected from 0, 1, 2 and 3; m is selected from 0, 1, 2and 3; up to 3 groups of Ring A selected from —CR₁═, —CR₂═ and —CR₅═ areoptionally replaced with —N═; R₁, R₂ and R₅ are independently selectedfrom hydrogen, hydroxy, halo, cyano, C₁₋₆alkyl,halo-substituted-C₁₋₆alkyl, hydroxy-substituted-C₁₋₆alkyl andcyano-substituted-C₁₋₆alkyl; R₃ and R₄, together with the carbon atomsto which R₃ and R₄ are attached, form a 5 to 6 member heterocycle fusedto ring A containing up to 4 radicals selected from O, C(O), S(O)₂,CR₁₁R₁₂ and NH; wherein each R₁₁ and R₁₂ are independently selected fromhydrogen, C₁₋₃alkyl, and halo-substituted-C₁₋₃alkyl; or R₁₁ and R₁₂,together with the carbon to which they are both attached, formsC₃₋₇cycloalkyl; R₆ and R₇ are independently selected from hydrogen,C₁₋₃alkyl and halo-substituted-C₁₋₃alkyl; or R₆ and R₇, together withthe carbon to which they are both attached, forms C₃₋₇cycloalkyl; R₈ isselected from C₁₋₆alkyl, C₂₋₆alkenyl, halo-substituted-C₁₋₆alkyl andhydroxy-substituted-C₁₋₆alkyl; or two R₈ groups can combine to form analkyl bridge; or when two R₈ groups are attached to the same carbonatom, they, together with the carbon to which they are both attached,form C₃₋₇cycloalkyl; R₉ is selected from L₁-C₆₋₁₀aryl,L₁-C₁₋₁₀heteroaryl, C₁₋₆alkyl, L₁-C₃₋₁₂cycloalkyl andL₁-C₃₋₈heterocycloalkyl; wherein said aryl, heteroaryl, cycloalkyl andheterocycloalkyl of R₉ can be optionally substituted with 1 to 3radicals independently selected from halo, cyano, hydroxy, C₁₋₃alkyl,halo-substituted-C₁₋₃alkyl, cyano-substituted-C₁₋₃alkyl,hydroxy-substituted-C₁₋₃alkyl, —C(O)R₁₃, —C(O)NR₁₃R₁₄; wherein each R₁₃and R₁₄ are independently selected from hydrogen and C₁₋₆alkyl; L₁ is abond, C₁₋₃ alkyl or halo-substituted-C₁₋₃ alkyl; Y is N or CR₁₀; R₁₀ isselected from hydrogen, C₁₋₆alkyl, —NR₁₅R₁₆, —NR₁₅C(O)R₁₆ and—C(O)NR₁₅R₁₆; wherein each R₁₅ and R₁₆ are independently selected fromhydrogen, C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₁₀heteroaryl, C₃₋₁₂cycloalkyl andC₃₋₈heterocycloalkyl; wherein said aryl, heteroaryl, cycloalkyl andheterocycloalkyl can be optionally substituted with 1 to 3 radicalsindependently selected from halo, hydroxy, cyano, C₁₋₆alkyl,halo-substituted-C₁₋₆alkyl, C₁₋₆ alkoxy and halo-substituted-C₁₋₆alkoxy;and the pharmaceutically acceptable salts thereof.
 2. The compound ofclaim 1, wherein: n is selected from 1 and 2; m is selected from 0, 1and 2; up to 3 groups of Ring A selected from —CR₁═, —CR₂═ and —CR₅═ areoptionally replaced with —N═ R₁, R₂ and R₅ are hydrogen; R₆ and R₇ arehydrogen; R₈ is selected from C₁₋₆alkyl, halo-substituted-C₁₋₆alkyl andhydroxy-substituted-C₁₋₆alkyl; or two R₈ groups can combine to form analkyl bridge; or when two R₈ groups are attached to the same carbon,they, together with the carbon to which they are both attached, formC₃₋₇cycloalkyl; R₉ is selected from L₁-C₆₋₁₀aryl, L₁-C₁₋₁₀heteroaryl,C₁₋₆alkyl, L₁-C₃₋₁₂cycloalkyl and L₁-C₃₋₈heterocycloalkyl; wherein saidaryl, heteroaryl, cycloalkyl and heterocycloalkyl of R₉ can beoptionally substituted with 1 to 3 radicals independently selected fromhalo, cyano, hydroxy, C₁₋₃alkyl, halo-substituted-C₁₋₃alkyl,cyano-substituted-C₁₋₃alkyl, hydroxy-substituted-C₁₋₃alkyl, —C(O)R₁₃,—C(O)NR₁₃R₁₄; wherein each R₁₃ and R₁₄ are independently selected fromhydrogen and C₁₋₆alkyl; L₁ is a bond or C₁₋₃alkyl; Y is CR₁₀, and R₁₀ ishydrogen.
 3. The compound of claim 2, wherein the 5 to 6 memberheterocycle fused to ring A formed from R₃ and R₄, together with thecarbon atoms to which R₃ and R₄ are attached, is selected from:


4. The compound of claim 3, wherein R₈ is selected from methyl, ethyl,trifluoromethyl, difluoromethyl, fluoromethyl and hydroxy-methyl; or twoR₈ groups can combine to form an alkyl bridge selected from methyl,ethyl and propyl; or two R₈ groups are attached to the same carbon,they, together with the carbon to which they are both attached, formcyclopropyl.
 5. The compound of claim 4, wherein R₉ is selected fromC₃₋₇cycloalkyl, C₄₋₇heterocycloalkyl, phenyl, pyridinyl, pyrazinyl,pyrimidinyl and furo[3,2-c]pyridin-4-yl; wherein said phenyl, pyridinyl,pyrazinyl, pyrimidinyl or furo[3,2-c]pyridin-4-ylis optionallysubstituted with 1 to 3 radicals independently selected fromtrifluoromethyl, cyano, bromo, chloro, hydroxy-methyl, methyl-carbonyl,methyl, amino-carbonyl, nitro, iodo, fluoro, methoxy-carbonyl, hydroxy,amino, carboxy and methoxy.
 6. The compound of claim 1 selected from:6-{4-[4-(5-trifluoromethyl-pyridin-2-yl)-[1,4]diazepan-1-ylmethyl]-1H-pyrazol-3-yl]-benzo[e][1,3]oxazine-2,4-dione;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((S)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3H-benzo[e][1,3]oxazine-2,4-dione;6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydrobenzo[e][1,3]oxazin-2-one;7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;7-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)isoquinoline-1,3(2H,4H)-dione;6-(4-44-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;7-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-1,4-diazepan-1-yl)methyl)-1H-pyrazol-3-yl)-1,2-dihydroisoquinolin-3(4H)-one;and6-(4-(((R)-4-(5-(trifluoromethyl)pyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydroquinazolin-2(1H)-one;(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;6-(4-43-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-5-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptan-6-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-42-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((3-(5-(trifluoromethyl)pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;(S)-6-(4-43-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one;6-(4-((4-(2,3-dimethylphenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one;(S)-6-(4-43-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-(trifluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-2-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)isonicotinonitrile;(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)pyrimidin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(5-methylpyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(5-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-2H-1,2,3-triazol-4-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-4,4-dimethyl-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;6-(4-((4-(5-(trifluoromethyl)pyridin-2-yl)-4,7-diazaspiro[2.5]octan-7-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(2-methyl-4-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)nicotinonitrile;(R)-6-(4-((4-(5-chloropyridin-2-yl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-1-((3-(2-oxo-3,4-dihydro-2H-benzo[e][1,3]oxazin-6-yl)-1H-pyrazol-4-yl)methyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-2-carboxylicacid;(S)-6-(4-((2-(hydroxymethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((2-(fluoromethyl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((4-(2-fluorobenzyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((4-(4-chlorophenyl)-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(R)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((3-methyl-4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)benzo[d]oxazol-2(3H)-one,and(R)-6-(4-((3-methyl-4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one;(S)-6-(4-((4-cyclohexyl-3-methylpiperazin-1-yl)methyl)-1H-pyrazol-3-yl)-3,4-dihydro-2H-benzo[e][1,3]oxazin-2-one.7. A method for modulating T and B lymphocyte development and functionin a subject for the treatment of autoimmune diseases, the methodcomprising administering to the subject a pharmaceutical compositioncomprising an effective amount of an agent which modulates the kinaseactivity or cellular level of an ITPKb molecule; thereby modulating Blymphocyte differentiation and function in a subject.
 8. The method ofclaim 7 wherein the agent down-regulates the cellular level of the ITPKbmolecule.
 9. The method of claim 8 wherein the agent is a compound ofclaim
 1. 10. The method of claim 9 wherein the agent inhibits the kinaseactivity of the ITPKb molecule.
 11. The method of claim 10 wherein thesubject is human and the ITPKb molecule is human ITPKβ.
 12. The methodof claim 11 in which the autoimmune disease is selected from rheumatoidarthritis, systemic lupus erythematosus, idiopathic thrombocytopenicpurpura, hemolytic anemia, and psoriasis.
 13. The method of claim 11wherein the subject suffers from B cell lymphoma.